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Molecular Architecture And Dynamics Of The Mammalian Secretory Pathway

- insights from the functional organeller proteomics studies

Xuequn Chen, Ph.D.

Department of Physiology School of Medicine

Wayne State University

Overview of organellar proteomics

EMBO Rep. 2006: 874-9

Exocrine pancreas, acinar cells and zymogen granules

Modified from Lodish et. al. Molecular Cell Biology

Jamieson, JD BBRC 1999

A working model for ZG biogenesis, trafficking and exocytosis

Tethering

SNARE

complex

ZG Golgi

Actin filaments

Budding

Apical membrane

Docking

Fusion

t-SNARE

Microtubule

motor proteins

Rab

v- SNARE

Transport

AIM - ZG proteomics study

• Individual components • The topology, copy numbers of ZGM proteins • Protein complexes on ZGM • Dynamic chagnes upon stimulation and in

diseases

Develop a quantitative model of ZGM molecular architecture and dynamics

Purification of ZG membrane

Rat pancreatic homogenates (~ 1 g)

S2 P2 (~100 mg)

highly purified ZGs (~20 mg)

Percoll gradient

nigericin

Lysis of ZGs

contents membrane (~1.2 mg)

purified membrane Na2CO3 wash

KBr wash

Highly purified membrane (~0.3 mg)

Mitochondria,

ER

Zymogen granules

Before centrifugation

After centrifugation

Incr

ease

d de

nsity

4 7 pH

2D gel map of Na2CO3-washed ZGM proteins SD

S-PA

GE

Localization of representative proteins in isolated acini

Localization of representative proteins on isolated ZGs

Summary: new insights in ZG proteins and their functional organizations

Tethering

Actin filaments

Docking

Fusion Golgi

Budding motor proteins

Rabs

SNAREs

Transport

Dynein/ Dynactin Myosin I Myosin Vc

Rab3D Rab6

Rab11A Rab27B

Rap1

Myosin Vc Slp 1 Slp 4 Noc 2

VAMP 2 VAMP 3 VAMP 8

Syntaxin 3 Syntaxin 7 SNAP 29

Protein-protein interaction network analysis of identified ZGM proteins

Dynein

Dynactin

V-ATPase

γ-secretase

SNAREs Rab/ Rab effectors

Global topology analysis of ZG membrane

iTRAQ: reagent design and workflow (PRG)

Inte

nsity

m/z

114

115

116

117

Peptide Quantification

Peptide Identification

m/z

Inte

nsity

Tryptic peptides

iTRAQ reagents

31 114

30 115

29 116

28 117

iTRAQ-labeled Peptide mixture

31 114 PRG +

30 115 PRG +

29 116 PRG +

28 117 PRG +

MS MS/MS

Isobaric tags for relative and absolute quantification

iTRAQ-based ZGM topology analysis

Proteinase K Control

ZGM ZGM Trypsin digestion

iTRAQ labeling

2D LC-MALDI-MS/MS

ZG lysis

Digestion (15 min, 4 °C)

tryptic peptides tryptic peptides

0’ 10’ 15’ 30’

Amylase

Rab3D

Pellet down

3430 MS/MS spectra 1079 peptides

≤ 1% false discovery rate

An example MSMS spectrum of iTRAQ-labeled peptide

9.0 254.4 499.8 745.2 990.6 1236.0Mass (m/z)

0102030405060708090

100

% In

tens

ity

117.1742

428.2780

72.1544 175.1547

112.1453742.500687.1511

643.4040411.2469115.1592 527.3760372.252459.1288 1048.6487232.1721 542.3517426.2951 685.4176258.194485.1325

117.17

116.16

115.15114.16

9.0 254.4 499.8 745.2 990.6 1236.0Mass (m/z)

0102030405060708090

100

% In

tens

ity

117.1742

428.2780

72.1544 175.1547

112.1453742.500687.1511

643.4040411.2469115.1592 527.3760372.252459.1288 1048.6487232.1721 542.3517426.2951 685.4176258.194485.1325

117.17

116.16

115.15114.16

9.0 254.4 499.8 745.2 990.6 1236.0Mass (m/z)

0102030405060708090

100

% In

tens

ity

117.1742

428.2780

72.1544 175.1547

112.1453742.500687.1511

643.4040411.2469115.1592 527.3760372.252459.1288 1048.6487232.1721 542.3517426.2951 685.4176258.194485.1325

117.17

116.16

115.15114.16

117.17

116.16

115.15114.16

117.17

116.16

115.15114.16

Peptide distributions on different iTRAQ ratios

0

50

100

150

200

250

0

0.15

0.3

0.45

0.6

0.75

0.9

1.05

1.2

1.35

1.5

1.65

1.8

1.95

iTRAQ ratios (CT/CT)

# of

pep

tides

0 10 20 30 40 50 60 70 80 90

100

0

0.15

0.3

0.45

0.6

0.75

0.9

1.05

1.2

1.35

1.5

1.65

1.8

1.95

iTRAQ ratios (PK/CT)

# of

pep

tides

control Vs.

control

Proteinase K Vs.

control

Peptide distributions on iTRAQ ratios (PK/CT)

0

10

20

30

40

50

60

70

80

90

100

0

0.15

0.3

0.45

0.6

0.75

0.9

1.05

1.2

1.35

1.5

1.65

1.8

1.95

iTRAQ ratios (PK/CT)

# of

pep

tides

Peptides with known topology

Cytoplasmic Dynactin 1 Dynein Myosin I Rab 1A Rab 2A Rab 3D Rab 5B Rab 11A Rap 1 VAMP 2 VAMP 8

Luminal Amylase Anionic trypsin 1 Carboxypeptidase A1 Carboxypeptidase A2 Chymotrypsinogen B Colipase Elastase 1 GP 2 GP 3 Itmap 1 Sterol esterase Syncollin ZG 16

0

5

10

15

20

25

30

0

0.1

5

0.3

0.4

5

0.6

0.7

5

0.9

1.0

5

1.2

1.3

5

1.5

1.6

5

iTRAQ ratio (PK/CT)

# of

pep

tides

Luminal

Cytoplasmic

Cytoplasmic and luminal peptides had distinct iTRAQ ratios

MSDFDSNPFADPDLNNPFKDPSVTQVTRNVPPGLDEYNPFSDSRTPPPGGVKMPNVPNTQPAIMKPTEEHPAYTQITKEHALAQAELLKRQEELERKAAELDRREREMQNLSQHGRKNNWPPLPSNFPVGPCFYQDFSVDIPVEFQKTVKLMYYLWMFHAVTLFLNIFGCLAWFCVDSSRAVDFGLSILWFLLFTPCSFVCWYRPLYGAFRSDSSFRFFVFFFVYICQFAVHVLQAAGFHNWGNCGWISSLTGLNKNIPVGIMMIIIAALFTASAVISLVMFKKVHGLYRTTGASFEKAQQEFATGVMSNKTVQTAAANAASTAATSAAQNAFKGNQM

A

B

C

Cytoplasm

Lumen

ZGM

D

Peptide position

Ion Score C.I. %

Exp. 1 PK/CT

Exp. 2 PK/CT

29-44 100 0.25 ± 0.07 0.12 ± 0.03

299-311 100 0.12 0.16 ± 0.07

312-334 93.1 0.23 NA

The topology model of SCAMP1

Molecular architecture of pancreatic zymogen granule

GTP

N

C

N

C

N C

SCAMP1-4

ENTP1

N

C

APP

Itmap1

N

C

GGTase

Rab3D Rab27B

N

C

Pantophysin

N

C pIgR

C

N VAMP2,8

C

N Syntaxin7,12

C

N

Cation-chloride cotransporter

N

C Presenilin2

N

C

Aminopeptidase N

N

C Nicastrin

Digestive Enzymes

GTP Rap1

MARCKS

ZG proteins and their copy numbers

GeLC-MRM strategy for ZG copy number determination

Protein of Interest

Amino Acid

Sequence Form

Precursor Ion

(monoisotopic)

SRM Ion

RAB 3D [24,34]

LLLIGNSSVGK

Native- Light 550.8373 648.3311

(y7)

RAB 3D [24,34]

LLLIGNSSVGK

Native- Light 550.8373 874.4993

(y9)

RAB 3D [24,34]

LLLIGNSSVGK

Native- Light 550.8373 761.4152

(y8)

RAB 3D [24,34]

LLLIGNSSVGK

Native- Light 550.8373 227.1754

(b2)

Development of MRM transitions for a unique Rab3D peptide

Protein of Interest

Amino Acid

Sequence Form

Precursor Ion

(monoisotopic)

SRM Ion

VAMP8 [14,23]

NLQSEVEGVK

Native- Light 551.7906 747.3883

(y7)

VAMP8 [14,23]

NLQSEVEGVK

Native- Light 551.7906 875.4469

(y8)

VAMP8 [14,23]

NLQSEVEGVK

Native- Light 551.7906 228.1343

(b2)

Development of MRM transitions for a unique VAMP8 peptide

Protein of Interest Amino Acid Sequence Form Precursor Ion

(monoisotopic) SRM Ion

VAMP8 [14,23] NLQSEVEGVK Native- Light 551.7906 875.4469 (y8)

VAMP8 [14,23] NLQSEVEGVK AQUA- Heavy 555.7977 883.4469 (y8)

RAB 3D [24,34] LLLIGNSSVGK Native- Light 550.8373 648.3311 (y7)

RAB 3D [24,34] LLLIGNSSVGK AQUA- Heavy 554.8444 656.3453 (y7)

RAB3D_[24,34] (554.8444++)/(550.8373++) Combined Chromatogram.

VAMP8_[14,23] (555.7977++)/(551.7906++) Combined Chromatogram

Absolute quantification of Rab3D and VAMP8 using AQUA peptides

(ng)

Quantification of Rab3D by WB using GST-Rab3D as internal standard

Rab3D

VAMP8

Immunofluorescence and bright field imaging of isolated ZGs

Determination of the size distribution of ZGs by EM and AFM

Secretory vesicles

Average size (nm) Copy numbers per vesicle Density (molecules /

µm2)

ZG 750 VAMP8: 6500 3678

Rab3D: 7400 4187

SV 45 VAMP2: 70 11003

Rab3A: 10 1572

ZG

SV

Comparison between pancreatic ZGs and neuronal SVs

Takamori et al. Cell 2006

Summary

A total of 231 proteins identified using multiple separation techniques

ZGM localization confirmed for some novel observations

including Rab11A, Rab27B, Rap1, SNAP 29, Myosin Vc and Slp 1

iTRAQ-based quantitative proteomics approach used to systematically ananlyze the enrichment and topology of ZG membrane proteins

Copy numbers of selected ZG proteins determined by LC-

MRM and AQUA peptides

Future directions

• ZG protein complexes by BN-PAGE, co-IP and

xlinking • Dynamic changes of ZG composition upon

stimulation and in diseases (e.g. pancreatitis)

Dr. Chen’s Lab • Dr. Jin-sook Lee • Patricia Skallos • Garrett Hubbs Dr. Jena’s Lab •Dr. Bhanu Jena

Acknowledgements

Dr. Stemmer’s Lab •Dr. Paul Stemmer •Dr. Joseph Caruso University of Michigan Dr. Philip Andrews’ lab Dr. John Williams’ lab

Fundings

Selection of Rab3D and VAMP8 for Absolute Quantification

Figure 5: RAB3D Scaffold View.

Figure 6: VAMP8 Scaffold View

Peptide position

Ion Score C.I. %

Exp. 1 PK/CT

Exp. 2 PK/CT

227-236 100 0.84 ± 0.10 0.93

341-354 100 0.63 ± 0.19 0.68 ± 0.20

738-754 100 0.32 ± 0.12 0.29 ± 0.10

764-778 100 0.12 ± 0.04 0.18 ± 0.01

MTSSPFLDPW PSKAVSIRER LGLGDRPNDS YCYNSAKNST VLQGVTFGGI PTVLLLDVGC FLVLILVFSI IRRKFWDYGR IALVSEAGSE ARFRRLSSSS SGQQDFESEL GCCSWLSAIF RLHDDQILEW CGEDAIHYLS FQRHIIFLLV VVSFLSLCVI LPVNLSGDLL DKDPYSFGRT TIANLQTDND LLWLHTVFSV IYLFLTVGFM WHHTRSIRYK EESLVRQTLF ITGLPREARK ETVESHFRDA YPTCEVVDVQ LCYSVAKLIN LCKERKKTEK SLTYYTNLQV KTGRRTLINP KPCGQFCCCE VQGCEREDAI SYYTRMNDSL TERITAEECR VQDQPLGMAF VTFREKSMAT FILKDFNACK CQGLRCKGEP QPSSYSRELC VSKWSVTFAS YPEDICWKNL SIQGVRWWLQ CLGINFSLFV VLFFLTTPSI IMSTMDKFNV TKPIHALNNP VISQFFPTLL LWSFSALLPT IVYYSTLLES HWTRSGENRI MVSKVYIFLI FMVLILPSLG LTSLDFFFRW LFDKTSSDTS IRLECVFLPD QGAFFVNYVI ASAFIGSGME LLRLPGLILY TFRMIMAKTA ADRRNVKQNQ AFEYEFGAMY AWMLCVFTVI MAYSITCPII VPFGLIYILL KHMVDRHNLY FAYLPAKLEK RIHFAAVNQA LAAPILCLFW LFFFSFLRLG LTAPATLFTF LVALLAILAC LLYTCFGCFK HLSPWNYKTE EPVGDKRNEA EAHAPPPFTP YVPRILNGLT SERTALSPQQ QQTYGAIRNI SGTLPGQLVA QDPSDTVAGV YQES

A

ZGM

Lumen

Cytoplasm

C B

The topology model of Tm63A

A mixture model for estimating peptide topology

Experiment #1 Experiment #2

iTRAQ ratio (PK/CT) iTRAQ ratio (PK/CT)

# of

pep

tides

# of

pep

tides

Green: experimental distribution Blue: model distribution – cytoplasmic Red: model distribution – luminal Dashed: training set distribution

y = 15128x R² = 0.8287

y = 8640.6x R² = 0.8798

0

5000

10000

15000

20000

25000

30000

35000

0 1 2 3

GST-Rab3D

BSA

Quantification of BSA std & GST-Rab3D using Image J

y = 8640.6x R² = 0.8798

0

5000

10000

15000

20000

25000

0 0.5 1 1.5 2 2.5 3

BSA BSA

Resuspend mouse ZGs in 160 ul H buffer

80 ul for SDS-PAGE 0.977 x 106 ZGs/ ul

80 ul for characterizing ZGs with different imaging approaches

ZG density by hemocytometer: 0.977 x 106 / ul

Add 80 ul lysis buffer, 4xSDS buffer etc to 210 ul (~ 2ug/ul protein)

0.372x 106 ZGs/ ul

Dilution factor 2.625

Run 50 ul ZG lysate on SDS-PAGE 0.372x 106 ZGs/ ul

Fixed

ZG size distribution: D = 750 ± 120 nm

Cut gel slices and in-gel digestion ?% recovery is unknown

For VAMP8 in band 2 & 3, resuspend tryptic peptide in 32ul solvent A and injected 5 ul for LC-MS/MS

dilution factor 6.4

AQUA: 5 fmole

AQUA: 50 fmole

VAMP8: 27.4 fmole for

2.91x 106 ZGs

VAMP8: 36 fmole for

2.91 x 106 ZGs

VAMP8: 6564 copies per ZG

For Rab3D in band 9, resuspend tryptic peptide in 108ul solvent A and injected 5 ul for LC-MS/MS

dilution factor 21.6

AQUA: 5 fmole

AQUA: 50 fmole

Rab3D: 10 fmole for

0.87x 106 ZGs

VAMP8: 11.5 fmole for

0.87 x 106 ZGs

VAMP8: 7439 copies per ZG

Based on “Counting 1”

The working principles of Blue native/SDS-PAGE

Plant Methods. 2005 Nov 16;1(1):11.

1.Itmap 1 2. Propionyl-CoA carboxylase α chain 3. Propionyl-CoA carboxylase β chain 4. Prohibitin 2 5. Prohibitin 6. Phosphodiesterase 3 7. NADP transhydrogenase 11. Ubiquitol cytochrome c-reductase 13. x 8. VATPase a1 subunit 12. VATPase V0 subunit d 14. VATPase V0 subunit c 10. Heat shock protein 65 15. ATPase β subunit 16. Glutamate dehydrogenase 18. GP2 17. x 19. X 20. X 22. X 21.Gamma glutamyl transpeptidase 23.Gamma glutamyl transpeptidase 24, 25. Syncollin 28. Elastase 2 31. Sterol esterase 32. GP3

Two dimensional separation of ZGM by BN/SDS-PAGE

1236

480

242

146

66

720

1048

KD

a

WB results from 2nd dimension BN/SDS-PAGE

Rab27B Rab3D Rap1 Syntaxin 3 VAMP 8 VAMP 2

KDa 720 480 242 146 66 20

GST-Rab3D affinity purification to identify interacting partners

250 150

100 75

50

37

25

20

15

10

KDa

GDP GTPγS

Myosin Vc (204 KDa)

Observed Delta Score Peptide 1286.60 0.00 26 K.LGSAEEFNYTR.M

1468.71 -0.00 31 R.LIQDSNQYQFGR.T

Trysin digestion

Measure peptide mass

List of measured peptide masses matched against theoretically predicted peptides of proteins in databases

m/z

Proteomics analysis leads to new hypotheses about ZG functions

• Rab27B localized to ZG membrane and plays a positive role in regulated exocytosis

• Two potential Rab interacting proteins identified and their interactions with Rab3D and Rab27B confirmed

Identification and confirmation of Rab27B on ZGM

A. Start - End Observed Mr(expt) Mr(calc) Delta Miss Sequence

12 - 22 1029.58 1028.57 1028.59 -0.01 0 LLALGDSGVGK 38 - 47 1168.64 1167.63 1167.63 0.00 0 FITTVGIDFR* 51 - 64 1383.66 1382.65 1382.63 0.02 0 VVYDTQGADGSSGK* 83 - 90 942.50 941.49 941.50 -0.00 0 SLTTAFFR 112 - 134 2678.21 2677.20 2677.26 -0.06 0 NWMSQLQANAYCENPDIVLIGNK 155 - 172 1974.93 1973.93 1973.94 -0.01 0 YGIPYFETSAATGQNVEK* 173 - 183 1277.70 1276.69 1276.69 -0.01 0 SVETLLDLIMK Oxidation(M)

B. 1 MTDGDYDYLI KLLALGDSGV GKTTFLYRYT DNKFNPKFIT TVGIDFREKR 51 VVYDTQGADG SSGKAFKVHL QLWDTAGQER FRSLTTAFFR DAMGFLLMFD 101 LTSQQSFLNV RNWMSQLQAN AYCENPDIVL IGNKADLLDQ REVNERQARE 151 LAEKYGIPYF ETSAATGQNV EKSVETLLDL IMKRMEKCVE KTQVPDTVNG 201 VNSGKVDGEK PAEKKCAC

total ZG ZG-cont ZG-memb

Rab27B

Rab27B plays an important role in acinar secretion

Am

ylas

e R

elea

se

(% o

f tot

al /

30 m

inut

es)

0

2

4

6

8

10

12

14

Gal WT QL NI Gal WT QL NI

*

*

basal CCK(300pM)

Proteomic analysis of ZG membrane helps to develop hypothesis on functional protein complex

Modified from Nat Cell Biol. 2002 Apr;4(4):271-8

Myosin Vc

ZG

Slp 1, Slp 4, ?

Rab27B/3D

Slp1 is localized on ZG membrane

LNcaP total PNS ZG ZG-cont ZG-memb

Slp1

A.

B.

Anti-Slp1 (red)

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